ABSTRACT Diabetic retinopathy remains the leading cause of blindness in working-age adults. Landmark clinical studies have documented that even after achieving and maintaining good glycemic control for many years, damage instilled by the prior poor glycemic control becomes difficult to undo, and the results have suggested that the prior hyperglycemia leaves a legacy. This `metabolic memory' phenomenon is also duplicated in vitro and in vivo experimental models of diabetic retinopathy. Termination of hyperglycemia in rats does not reverse mitochondrial dysfunction and DNA (mtDNA) damage, DNA repair enzyme MutL homolog 1 (Mlh1) remains subnormal, and impaired mtDNA transcription continues to compromise the electron transport chain (ETC). Stability of both genomic and structure/physiology are important for mitochondrial homeostasis; mitochondrial fusion enzyme mitofusin 2 (Mfn2) also remains subnormal even after cessation of hyperglycemia. Recent studies have documented that genomic functions are also modulated by epigenetic modifications, the modifications that regulate gene expression without changing the DNA sequence. Our recent research has shown that diabetes activates DNA methylation machinery in the retina and its capillary cells, and this activation is not terminated by reversal of hyperglycemia. Thus, the central hypothesis is that due to sustained epigenetic modifications, mitochondrial DNA and structure/function remain damaged. Dysfunctional mitochondria continues to fuel into the vicious cycle of free radicals, and cessation of hyperglycemia fails to arrest the progression of incipient diabetic retinopathy. Aim 1 will investigate the role of epigenetic modification in mitochondrial genomic stability. Our model predicts that due to sustained Mlh1 promoter DNA hypermethylation, mitochondrial genomic stability remains compromised, and impaired mtDNA transcription continues to damage ETC system, fueling into mitochondrial damage. Aim 2 will determine how epigenetic modifications regulate mitochondrial structural/physiological homeostasis, and will investigate the role of epigenetic modifications of Mfn2 promoter in continued mitochondrial damage. Aim 3 will determine the effect of protection of mitochondrial homeostasis in the resistance of diabetic retinopathy to halt by directly inhibiting epigenetic modifications during normal glycemia, which has followed hyperglycemia. The plan will employ in vitro (retinal endothelial cells) and in vivo (retinal microvessels from rodents maintained in varied glycemic control) models of metabolic memory, and will utilize fully optimized molecular biological and pharmacological approaches. Our overall goal is to understand the molecular mechanism responsible for continued mitochondrial damage in the progression of diabetic retinopathy. The proposal is based on a testable central hypothesis, and these innovative studies carry a significant translational impact as they are expected to define the role of epigenetics in continued mitochondrial damage, and identify novel therapeutic targets to inhibit the progression of this sight-threatening disease.